The long term objective of this project is to determine the importance of pulsatile delivery of gonadotropins to the development and function of rat ovarian cells. This objective is being approached through use of a perfusion system that can mimic closely conditions in vivo, exert precise control of gonadotropic input to cultured ovarian cells, and monitor the responses of the cells to the applied input signals. The perfusion system will be used to study the effects of providing trains of gonadotropic pulses to granulosa cells and intact follicles. Pulse trains to be studied will be ones that closely mimic those observed in the intact rat, in terms of frequency, amplitude and shape. Specifically it is proposed to test: if pulsatile gonadotropins have control advantages over constant concentrations of gonadotropins with amplitude and frequency being inversely related; if each pulse of gonadotropin induces a degree of refractoriness that is related in some fashion to the concentration/duration of the applied gonadotropic pulse if for maximally effective restimulation, the magnitude and duration of this refractoriness dictate the optimal time and amplitude of a subsequent gonadotropic pulse; and if these stimulus-response relationships differ for different ovarian cell types, are affected by prior cellular development or are modulated by steroids and other hormones/factors acting in a paracrine fashion. Response parameters to be measured include steroid secretion (by solid phase immunoassay), release of protons and electroactive molecules, presumably ascorbic acid (by ion selective potentiometry and amperometric cyclic voltammetry), and morphology (by light and electron microscopy). When the envisioned experiments are completed important information regarding gonadotropin amplitude-frequency relationships in controlling follicular function and development should be at hand. This includes obtaining a better understanding both of some of the intraovarian events operating in a paracrine fashion and of the nature of granulosa-the ca cell interactions that extend beyond provision of steroidogenic substrate and product. In addition, a better understanding should be obtained of the role that desensitization plays in determining these response relationships, whether or not each physiological pulse is associated with some degree of desensitization, and whether or not the resulting refractoriness is related to the size of the stimulating pulse. The analyzed and interpreted results should help investigators to decode trains of gonadotropin pulses: recognize and separate important from non-important information, and decipher why similar trains appear to, act differently to control the development and function of ovarian follicles and their contained granulosa cells.